The rate-limiting step in reverse cholesterol transport from macrophages is the hydrolysis of cholesteryl esters to free cholesterol, which is subsequently transported out of the cell to cholesterol acceptors. In human macrophages, the enzyme responsible for cholesteryl ester hydrolysis is carboxylesterase 1 (CES1). The overall hypothesis of parent grant R15 ES015348 was that exposure to bioactive metabolites of organophosphate (OP) insecticides will inhibit the ability of CES1 to hydrolyze cholesteryl esters, thus slowing the reverse cholesterol transport process and increasing the risk of atherosclerosis. Our published data, obtained during the current funding cycle, shows that CES1 can be inhibited by treatment with paraoxon, the active metabolite of the OP insecticide parathion, and that this inhibition can cause augmented accumulation of cholesteryl esters in human THP-1 macrophages (Crow et al., 2008). Moreover, our recent preliminary data show that recombinant CES1 and CES2 can hydrolyze the endocannabinoid compound 2-arachidonoylglycerol (2-AG) and its COX-2 derived prostaglandin-like metabolites (termed prostaglandin glyceryl esters or PG-Gs). Pretreatment of human THP-1 monocytes and macrophages with paraoxon can block the subsequent degradation of exogenously added 2-AG and PG-Gs. These are novel findings that suggest that exposure of CES1- and/or CES2-expressing cells to bioactive OP metabolites (e.g., paraoxon) will perturb the endocannabinoid tone in tissues composed of these cells. It has become increasingly clear that endogenous cannabinoids, which bind to the same class of receptors as the psychoactive component of marijuana (?9-THC), have important functions in health and disease. Therefore, we hypothesize that the endocannabinoid tone of vessel wall macrophages will be significantly perturbed by chronic exposure to bioactive OP metabolites and an activated endocannabinoid system can modulate cholesterol metabolism in macrophages. To explore this possibility, we propose to extend the scope of R15 ES015348 by studying the endocannabinoid system in a model cell culture system (macrophage foam cells) that we already use in the parent grant. To test our hypothesis we propose two aims: (1) Determine if combined treatment of cultured human THP-1 macrophages with oxidized (ox)LDL and bioactive metabolites of OP pesticides modulate components of the endocannabinoid system, including cannabinoid (CB) receptor levels, endocannabinoid biosynthetic enzymes, endocannabinoid catabolic enzymes (MAGL and CES1), and the levels of endocannabinoids themselves (2-AG and AEA). (2) Determine if endocannabinoids (2-AG and AEA) modulate cholesterol metabolism and efflux from human macrophages via a CB1- or CB2-mediated mechanism. PUBLIC HEALTH RELEVANCE: Atherosclerosis is the number one killer in the United States. The underlying pathophysiology of atherosclerosis is complex and multifactorial. One critical factor in the development/progression of atherosclerosis is environmental factors, such as toxicants, that may accelerate disease. Our research seeks to examine whether organophosphate (OP) pesticides are etiologically associated with atherosclerosis. Our research indicates that there are enzymes that regulate cholesterol metabolism, which can be inhibited by bioactive metabolites of OP pesticides. This competitive supplement will allow us to study these mechanisms in greater detail.